The long-term goal of this project is to test an innovative hypothesis regarding the role of macroglial M[unreadable]ller cells in the early development and progression of diabetic retinopathy. We hypothesize that M[unreadable]ller cells are a primary cellular target of diabetic insult and may play a pivotal role in the early stages of development and progression of diabetic retinopathy via alterations of purinergic receptor expression and function. Because M[unreadable]ller cells play a central role in coordinating the activity of neuronal cells and endothelial cells via purinergic signaling, such alterations within the M[unreadable]ller cells would predispose the retina toward neuronal dysfunction and loss as well as breakdown of the blood-retinal barrier and alterations in endothelial cell function. We will test this hypothesis by comparing the M[unreadable]ller cell profiles of P2X and P2Y purinergic receptor expression and functional profiles in nondiabetic and streptozotocin-induced diabetic rats at various time points following the onset of hyperglycemia which precede the time required for neuronal and vascular complications to develop in this animal model. Immunohistochemical analysis of retinal sections and Western blot analyses of retinal protein lysates will be used to compare protein levels, while RT-PCR analyses will be used to compare mRNA levels. We will also use the fluorescent calcium reporter fura-2 in optical imaging studies to compare the P2X- and P2Y receptor-mediated intracellular calcium dynamics of M[unreadable]ller cells from diabetic and nondiabetic rats at these same early time points. If alterations are observed in the P2X and/or P2Y receptors at these early time points, it will establish the M[unreadable]ller cell as a cellular target and the purinergic receptors as a molecular target for developing novel intervention and prevention strategies to combat the widespread loss of vision due to irreversible loss of neurons and breakdown of the blood-retinal barrier in late stages of diabetic retinopathy. We also hypothesize that the diabetes-induced changes in M[unreadable]ller cell purinergic receptor expression and function reflect a direct response of the M[unreadable]ller cells to diabetic conditions and are not merely consequences of other subtle diabetes-induced changes in the functioning of the retinal circuit. To test this hypothesis, we will employ an immortalized rat M[unreadable]ller (rMC-1) cell line to compare the expression and functional profiles of P2X and P2Y receptors in rMC-1 cells cultured in nondiabetic (low glucose and high insulin levels) with those of cells cultured in diabetic (high glucose and low insulin levels) culture media. Recapitulation of the changes noted in the diabetic rat model will establish the rMC-1 cell line as a useful model to determine the molecular mechanisms underlying the changes and to test the ability of both existing and novel therapeutic reagents to reverse or prevent these alterations. In summary, the combined proposed studies promise to provide a novel and rapid path toward identifying earlier intervention and prevention of blinding retinopathy in diabetic patients. Retinal disease often develops and progresses to blindness in many diabetic patients. While great strides have been made using drugs to slow down the progression of the later retinal complications of diabetes, less progress has been made toward identifying effective strategies for early intervention and prevention of diabetes-induced retinal damage. The proposed research will rapidly provide informative data regarding novel potential targets for early intervention and prevention of irreversible retinal damage caused by diabetes. [unreadable] [unreadable] [unreadable]